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Periodic Report Summary 1 - TINTIN (Training in neurodegeneration, therapeutics intervention and neurorepair)

Project title: Training in neurodegeneration, therapeutics intervention and neurorepair
Acronym: TINTIN
Funding scheme: FP7-MC-ITN
Co-Ordinator: Prof Gavin Davey, Trinity College Dublin, Dublin 2, Ireland
Contact Details: Telephone +353 18968408 Email gdavey@tcd.ie
Project web address http://www.tintin-itn.net/

Summary description of the project objectives
Dopamine producing neurons play a central role in major illnesses, such as anxiety and mood disorders, schizophrenia, autism-spectrum disorders, Parkinson’s disease, epilepsy, and dementia.
Discovery of the biochemical mechanisms that underlie dopamine neuron dysfunction will be enhanced by a researchers trained with multidisciplinary skillsets.
The overall objective of the TINTIN network is to:
• Train ESRs and ERs in multidisciplinary aspects of research related to normal and abnormal function of the dopaminergic neuron so that new research discoveries will lead to improved therapies for cohorts of European patients in which dopaminergic systems are at the basis of their disorder.
The scientific and technological objectives of TINTIN use interdisciplinary approaches to:
• Discover how autophagy in the dopamine neuron is related to lysosomal and mitochondrial dysfunction.
• Discover how novel genetic mutations in glycolipid and ganglioside metabolism relate to dopamine neuron degeneration.
• Utilize pluripotent stem cell technologies for studying dopaminergic neurodegeneration.
• Utilize computational and molecular design techniques to identify novel aspects of the neurodegenerative processes that may be selected as therapeutic targets.
• Identify and validate novel glycan-based biomarkers for use in clinical trials.

Description of the work performed since the beginning of the project
ESR1 – Modulation of antioxidant status in dysfunctional dopaminergic neurons: (i) In order to study the physiological role of endogenous astrocytic mitochondrial reactive oxygen species (mROS), mROS was down-modulated in glial cells using a transgenic mouse model. (ii) These outcome of these results show that glial mROS have a signaling role in astrocytes and promote neuronal antioxidant defenses. (iii) Currently, in vivo studies are investigating how physiological astrocytic mROS regulates neuronal redox status and function.
ESR2 - Characterisation of the redox status inside dopamine neurons using conditional transgenic mouse models: (i) In order to investigate in vivo brain cell metabolism, a long-term primary culture of neurons has been established using an oxygen-controlled incubator chamber. (ii) It was found that these cells, when incubated at different oxygen concentration, adapt their mitochondrial energy metabolism giving rise to significant changes in mitochondrial membrane potential, neuronal apoptosis, mitochondrial reactive oxygen species production, glycolysis and mitochondrial shape when compared with cells incubated under atmospheric oxygen conditions.
ESR3 - The effect of glutathione on mitochondrial dynamics & function in the dopamine neuron: (i) An investigation of the toxicity of alpha-synuclein, both wild type and mutations A53T, A30P and E46K on mitochondrial function in brain was conducted. Results show a preferential toxicity on complex I in brain mitochondria. (ii) Regulating the redox potential of mitochondria also showed differential sensitivity of brain mitochondria to toxins.
ESR4 - How does the cytochrome P450 system control drug metabolism in the neuron?: (i) In order to identify CYP expression in undifferentiated neuroblastoma SH-SY5Y cells were treated with various CYP inducers and analysed by qRT-PCR. Induction of the P450 isoforms 2D6, 2E1, and 1A1 were identified. (ii) Currently the role of CYP expression on neurotoxin sensitivity is being studied.
ESR5 – Crosstalk between mitochondrial & lysosomal dysfunction in the dopamine neuron: (i) Regulating glucocerebrosidase activity in SH-SY5Y cells and the effect on mitochondrial function is a major component of this project. GBA inhibition was found to regulate mitochondrial membrane potential. (ii) For future experiments, mitochondrial-lysosomal interactions in these cells will be studied by knocking down molecular motor proteins.
ESR6 – How does glucocerebrosidase control autophagy in the dopamine neuron?: (i) Pharmacological neuronal cell model of GBA1 deficiency revealed effects on biogenic amine turnover. (ii) In collaboration with the ER2 in UCL knock down GBA1 SH-SY5Y cells is being assessed. Glycolipid profiles in CSF from patients with low HVA are being studied in collaboration with ESR12 in NIBRT.
ESR7 - Human wild type and parkinsonian iPS cell lines: (i) In neuronally derived cells, removal of chemically damaged mitochondria does not seem to rely on the autophagy nucleation and initiation machinery, and does depends on the ubiquitin proteasome system. (ii) A stable iPS cell line has been developed, and is currently being differentiated in order to carry out similar experiments to investigate autophagy and mitophagy.
ESR8 - Neurotoxin models of Parkinson’s disease: (i) MPP+ treatment of dopamine and serotonergic stem cell derived neurons results in a misbalance between fusion and fission processes in mitochondria. (ii) MPP+ acts differentially on dopaminergic and serotonergic neurons derived from mESC. The mechanisms that underlie this differential sensitivity are under investigation.
ESR9 - In silico reconstruction of the dopamine transporter and design of new drugs with neuroprotective properties: (i) By performing homology modeling and molecular dynamic (MD) simulations of human dopamine transporter (hDAT) we obtained 3D structure models of hDAT in complex with dopamine (natural substrate), amphetamine (substrate – psychostimulant), cocaine (inhibitor) and modafinil (atypical inhibitor) in environment of lipid membrane and water. Results provided an understanding of the differences in binding and interactions of different compounds with the transporter. (ii) Building pharmacophore models of DAT substrates and inhibitors lead us to understand the chemical features that a compound needs to have in order to be substrate/inhibitor. This model will be used for docking of novel molecules to check suitability as antagonists for dopamine transporter.
ESR10 - Development of new drugs to inhibit the neurodegenerative process in dopamine neurons: (i) A method is being developed in which SHSY5Y cells can be used to detect lipid variance in PD and related diseases. The cells’ lipid profile was obtained using a newly developed lipid extraction method and Q-TOF LC/MS analysis. Preliminary trials suggest that the new lipid extraction method provides satisfactory recovery of lipids of cell samples compared to the gold standard methods. (ii) The toxicity of various neurotoxins will be investigated on undifferentiated and differentiated cells and the lipid profiles of toxin-treated cells will be compared with untreated cells, as well as those treated with potentially neuro-protective compounds, allowing identification of lipid biomarkers for the disease and development of neuro-protective drugs.
ESR11 - Characterisation of the cell surface glycome on the dopamine neuron using glycochip technology: (i) Development of MALDI-ToF-based platform to study relevant interactions between (neo)glycolipids and trimming enzymes. (ii) Development of a high-throughput computer aided processing method to compare proteomics data.
ESR12 - Glycans, glycolipids and gangliosides related to dopaminergic cell death?: (i) MS based method for glycan quantitation has been developed and submitted for publication. These methods will be used to characterize serum and cerebrospinal fluid N-glycans and reveal their biomarker potential in Parkinson’s disease. Serum samples are already prepared for LC- and CE-MS analysis. (ii) Future aims include revealing the possible correlations between glycosylation changes and disease progression in Parkinson’s disease using in vitro neuronal models.
ER1 - The mechanisms that control mitochondrial dynamics and function in the dopamine neuron: (i) This project will focus on the characterization of the role of monoamine oxidases (MAO) in the mitochondrial dynamics of Parkinson's disease-derived dopaminergic neurons. (ii) MAO activity levels in differentiated PC12 cells and cortical neurons have been characterized. Currently, the differentiation of human PD-iPS cells into dopamine neurons is being completed for further investigation of the effects of MAO on cell function.
ER2 - What mechanisms control autophagy in the dopamine neuron?: (i) Interference RNAs (shRNA) were designed and developed to silence the human GBA gene through lentiviral transduction of several human cell lines. (ii) The effects of GBA knockdown on neuronal function are being studied through HPLC quantification of metabolites involved in the dopamine pathway in neuronal cells differentiated to dopaminergic neurons.

Expected final results and their potential impact and use: There has been considerable success at the individual research project level, resulting in novel mechanistic aspects of brain cells being uncovered. These initial findings are being extended into neurons of the dopaminergic phenotype as well as into the biomarker arena for conditions involving dopaminergic dysfunction. The TINTIN consortium anticipates these discoveries to be extended as a direct result of continuous project integration. During the second half of the project high-impact scientific publications are expected as well as the generation of new putative anti-neurodegenerative therapeutics and intellectual property.

Reported by

THE PROVOST, FELLOWS, FOUNDATION SCHOLARS & THE OTHER MEMBERS OF BOARD OF THE COLLEGE OF THE HOLY & UNDIVIDED TRINITY OF QUEEN ELIZABETH NEAR DUBLIN
Ireland

Subjects

Life Sciences
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